Ionizing radiation (IR) can cause to significant tumor cell death, however resistance to radiotherapy can limit its efficacy. IR induces DNA double-strand breaks (DSBs) that can lead to toxic chromosomal rearrangements; such that limiting these rearrangements may be critical for radioresistance. Such IR-induced chromosomal rearrangements likely form via end joining (EJ) that uses incorrect DSB ends during repair. Thus, our long-term goal is to define the factors that limit rearrangements during EJ, and thereby develop therapeutic targets for tumor radiosensitization. To advance this goal, we developed a unique assay to quantify the use of correct versus incorrect ends during EJ of multiple DSBs. Using a chromosomal reporter with two tandem DSBs, and our technical innovation of generating site-specific non-cohesive DSBs, we can measure EJ that uses proximal ends that flank a single DSB versus EJ that uses distal ends of two DSBs. Distal end use during EJ is incorrect, since it causes a deletion rearrangement. Using this system, we found that the DNA damage response factors RAD50 and DNA-PKcs are important to limit incorrect end use during EJ. Since these factors also promote radioresistance, we propose to test our central hypothesis that the role of RAD50 and DNA-PKcs in limiting incorrect end use during EJ is critical for cellular radioresistance. A corollary of this hypothesis is that the funcion of these factors in limiting incorrect end use during EJ is a target for radiosensitization. Aim 1: To determine the importance to radioresistance of RAD50 function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. For this, we will examine a series of RAD50 mutants for the ability to complement a set of DNA repair functions, and promote radioresistance, in RAD50-deficient human cells. Aim 2: To determine the importance to radioresistance of DNA-PKcs function in limiting incorrect end use during EJ, as compared to its other roles in DNA repair. Using a similar approach as Aim 1, we will examine a series of DNA-PKcs mutants for the ability to complement DNA repair functions, and promote radioresistance, in DNA-PKcs-deficient mammalian cells. Aim 3. To determine how increasing the distance between two tandem DSBs affects incorrect end use during EJ, and the relative requirement of RAD50 and DNA-PKcs for limiting such EJ-mediated deletion rearrangements. These proposed studies are significant, because they will provide novel insight into how RAD50 and DNA-PKcs promote radioresistance, which is critical for their development as therapeutic targets for tumor radiosensitization. Our study is innovative because it will establish a new paradigm for understanding the role of RAD50 and DNA-PKcs in promoting radioresistance, and because we propose in Aim 3 to develop a new reporter system to examine how the distance between DSBs affects end use during EJ. Our proposed study is also innovative because it will provide insight into the importance of incorrect end use during EJ for radiation toxicity, which will lead to new therapeutic strategies for tumor radiosensitization.